This invention relates to recombinant anti-sickling hemoglobins suitable for use as therapeutics for the treatment of sickle cell anemia.
The gene that encodes hemoglobin S (the defect leading to sickle cell anemia) is inherited as an autosomal trait and occurs in the heterozygous condition as the sickle trait in 8-10% of black persons in the Unites States. Two major clinical features characterize sickle cell anemia: (1) chronic hemolysis that is stable and only moderately debilitating, and (2) acute, episodic vaso-occlusive crises that cause organ failure and account for most of the mortality and morbidity associated with the disease.
The molecular basis for sickle cell disease is an A to T transversion in the 6th codon of the human .beta.-globin gene. This simple transversion changes a polar glutamic acid residue to a non-polar valine (Ingram et al., Nature 178:792, 1956; Ingram et al., Nature 180:326, 1957) in the .beta.-globin polypeptide and, thus, drastically decreases the solubility of this hemoglobin (termed Hb S). When the intracellular concentration of Hb S is high and the partial pressure of oxygen is low in the capillary beds, the non-polar valine, which is on the surface of the hemoglobin molecule, interacts with two other non-polar residues on the surface of a second hemoglobin molecule, and initiates aggregation (Padlan et al., J. Biol. Chem. 260:8280-8291, 1985; Wishner et al., J. Mol. Biol. 98:179-194, 1975). Once approximately 10 hemoglobin monomers interact, long polymers rapidly accumulate, and complex 14-stranded fibers are formed (Crepeau et al., Nature 274:616-617, 1978; Dykes et al., J. Mol. Biol. 130:451-472, 1979; Eaton et al., Blood 70:1245-1266, 1987; Hofrichter et al., Proc. Natl. Acad. Sci USA 71:4864-4868, 1974). The formation of these fibers reduces the flexibility of red blood cells and leads to the occlusion of small capillaries. Intracellular fiber formation also results in erythrocyte membrane damage and increased red cell lysis (Noguchi et al., Blood 58:1057, 1981; Brittenham et al., Blood 65:183, 1985). The ensuing disease is characterized by a chronic hemolytic anemia with episodes of severe pain, and tissue damage that can result in stroke, kidney failure, heart disease, infection, and other complications (Bunn et al., Hemoglobin: Molecular, Genetic, and Clinical Aspects. (W. B. Saunders, Philadelphia, 1986)).